1. Field of the Invention
This invention relates to catecholamine receptors from mammalian species and the genes corresponding to such receptors. Specifically, the invention relates to the isolation, cloning and sequencing of complementary DNA (cDNA) copies of messenger RNA (mRNA) encoding a novel mammalian catecholamine receptor gene. The invention also relates to the construction of recombinant expression constructs comprising cDNA of this novel catecholamine receptor gene, said recombinant expression constructs being capable of expressing catecholamine receptor protein in cultures of transformed prokaryotic and eukaryotic cells. Production of the receptor protein in such cultures is also provided. The invention relates to the use of such cultures of such transformed cells to produce homogeneous compositions of the novel catecholamine receptor protein. The invention also provides cultures of such cells producing this catecholamine receptor protein for the characterization of novel and useful drugs. Antibodies against and epitopes of this novel catecholamine receptor protein are also provided by the invention.
2. Background of the Invention
Catecholamines are a class of naturally-occurring amino acid derivatives having a variety of physiological effects in the peripheral and central nervous systems. The parent compound is xcex2-phenylethylamine, and the catecholamines are derivatives of this parent compound. In addition to the naturally-occurring members of the class (epinephrine, norepinephrine and dopamine), a large number of synthetic compounds having biological activity have been developed and have utility as drugs (including albuterol, isoproterenol, propranolol, phenylephrine, amphetamine and methamphetamine). In the periphery, catecholamines are released by the sympathetic nervous system and adrenal medulla and are involved in integrating physiological responses to stress, while in the central nervous system the catecholamines constitute some of the most important neurotransmitter systems.
The effect of catecholamines are mediated through their receptors and their associated cell signaling systems (reviewed in Hoffman and Lefkowitz, 1982, Ann. Rev. Physiol. 44: 475-484; Civelli et al., 1993, Ann. Rev. Pharm. and Tox. 33: 281-307). These receptors are located in the plasma membrane of catecholamine-sensitive cells. Structurally, they are characterized by having a pattern of seven transmembrane domains (see,for example, U.S. Pat. Nos. 5,422,265,5,569,601, 5,594,108,5,883,226, 5,880,260,5,427,942 and 5,686,573). Functionally, certain of these receptors interact with adenylate cyclase, either stimulating or inhibiting the production of cyclic AMP thereby. These receptors include the adrenergic receptors (the a-1, a-2, b-1, b-2, and b-3 adrenergic receptors) and the dopamine receptors (the D1-, D2-, D3-, D-4-, and D5-dopamine receptors).
For example, epinephrine (adrenaline) and norepinephrine, as well as synthetic agonists of these catecholamines which mimic their biological functions, and antagonists which block these biological functions, exert their effects by binding to specific recognition sites (membrane receptors) situated on the cell membranes in the peripheral nervous system, Two principal classes of adrenergic receptors have been defined, the alpha-adrenergic receptors and the beta-adrenergic receptors. Five subtypes of adrenergic receptors ( a-1, a-2, b-1, b-2, and b-3 adrenergic receptors) have now been distinguished. The genes encoding these receptors have been isolated and identified (Cotecchia et al., 1988, Proc. Natl. Acad. Sci. USA 85: 7159-7163; Kobilka et al., 1987, Science 238: 650-656; Frielle et al., 1987, Proc. Natl. Acad. Sci. USA 84: 7920-7924; Emorine et al., 1987, Proc. Natl. Acad. Sci. USA 84: 6995-6999; Emorine et al., 1989, Science 245: 1118-1121). Analysis of these genes has made it possible to recognize that they belong to a family of integral membrane receptors exhibiting some homology (Dixon et al., 1998, Annual Reports in Medicinal Chemistry, 221-223; Emorine et al., 1988, Proc. NATO Adv. Res. Workshop), especially at portions of the seven transmembrane regions that are coupled to regulatory proteins, called G proteins, capable of binding molecules of guanosine triphosphate (GTP).
These membrane receptors, after they have bound the appropriate ligand (agonist or antagonist), are understood to undergo a conformational change that induces an intracellular signal that modifies the behavior of the target cell. Beta-adrenergic receptors catalyze the activation of a class of G proteins which in turn stimulates the activity of adenylate cyclase when they bind with catecholamine agonists, whereas alpha-adrenergic receptor antagonists act in competition with the agonists for the binding to the receptor and prevent the activation of adenylate cyclase. When adenylate cyclase is activated, it catalyses the production of an intracellular mediator or second messenger, especially cyclic AMP.
In the central nervous system, dopamine is a catecholamine neurotransmitter modulates neuronal cells involved in movement initiation, appetitive behavior, hormone release, and visual dark adaptation. In the periphery dopamine plays a role in modulating blood pressure and renal function (see generally Cooper et al., 1978, The Biochemical Basis of Neuropharmacology, 3d ed., Oxford University Press, New York, pp, 161-195). The diverse physiological actions of dopamine are in turn mediated by its interaction with a family of distinct dopamine receptors subtypes that are either xe2x80x9cD1-likexe2x80x9d or xe2x80x9cD2-like,xe2x80x9d which respectively stimulate and inhibit the enzyme adenylate cyclase (Kebabian and Calne, 1979, Nature 277: 93-96). Alterations in the number or activity of these receptors may be a contributory factor in disease states such as Parkinson""s disease (a movement disorder) and schizophrenia (a behavioral disorder) and attention deficit hyperactivity disorder (ADHD).
A great deal of information has accumulated regarding the biochemistry of the D1 and D2 dopamine receptors, and methods have been developed to solubilize and purify these receptor proteins (see Senogles et al., 1986, Biochemistry 25: 749-753; Sengoles et al., 1988, J. Biol. Chem. 263: 18996-19002; Gingrich et al., 1988, Biochemistry 27: 3907-3912). The D1 dopamine receptor in several tissues appears to be a glycosylated membrane protein of about 72 kD (Amlaiky et al., 1987, Mol. Pharmacol. 31: 129-134; Ninzik et al., 1988, Biochemistry 27: 7594-7599). The D2 receptor can also be glycosylated and has been suggested to have a higher molecular weight of about 90-150 kD (Amlaiky and Caron, 1985, J. Biol. Chem. 260: 1983-1986; Amlaiky and Caron, 1986, J. Neurochem. 47: 196-204; Jarvie et al., 1988, Mol. Pharmacol. 34: 91-97).
Dopamine receptors are primary targets in the clinical treatment of psycho-motor disorders such as Parkinson""s disease and affective disorders such as schizophrenia (Seeman et al., 1987, Neuropsychopharm. 1: 5-15; Seeman, 1987, Synapse 1: 152-333). Five different dopamine receptor genes (D1, D2, D3, D4 and D5) and various splice variants of their transcripts have been cloned as a result of nucleotide sequence homology which exists between these receptor genes (Bunzow et al., 1988, Nature 336: 783-787; Grandy et al., 1989, Proc. Natl. Acad. Sci. USA 86: 9762-9766; Dal Toso et al., 1989, EMBO J. 8: 4025-4034; Zhou et al., 1990, Nature 346: 76-80; Sunahara et al., 1990, Nature 346: 80-83; Sokoloff et al., 1990, Nature 347: 146-151; Civelli et al., 1993, Annu. Rev. Pharmacol. Toxicol. 33: 281-307; Van Tol et al., 1991, Nature 350: 610-4).
Catecholamine receptors are also targets for a host of therapeutic agents for the treatment of shock, hypertension, arrhythmias, asthma, migraine headache, and anaphylactic reactions, and include antipsychotic drugs that are use to treat schizophrenia and xcex2-blockers used to control high blood pressure.
The importance of catecholamine receptors, particularly in the brain and central nervous system, has created the need for the isolation of additional catecholamine receptors for the development of therapeutic agents for the treatment of disorders, including disorders of the CNS and most preferably treatment of disorders on mental health such as psychosis, in which catecholamines and their receptors have been implicated. There is also a need for developing new tools that will permit identification of new drug lead compounds for developing novel drugs. This is of particular importance for psychoactive and psychotropic drugs, due to their physiological importance and their potential to greatly benefit human patients treated with such drugs. At present, few such economical systems exist. Conventional screening methods require the use of animal brain slices in binding assays as a first step. This is suboptimal for a number of reasons, including interference in the binding assay by non-specific binding of heterologous (i.e., non-receptor) cell surface proteins expressed by brain cells in such slices; differential binding by cells other than neuronal cells present in the brain slice, such as glial cells or blood cells; and the possibility that putative drug binding behavior in animal brain cells will differ from the binding behavior in human brain cells in subtle but critical ways. The ability to synthesize human catecholamine receptor molecules in vitro would provide an efficient and economical means for rational drug design and rapid screening ofpotentially useful compounds. For these and other reasons, development of in vitro screening methods for psychotropic drugs has numerous advantages and is a major research goal in the pharmaceutical industry.
The present invention relates to the cloning, expression and functional characterization of a mammalian catecholamine receptor gene. The invention comprises nucleic acids having a nucleotide sequence of a novel mammalian catecholamine receptor gene. The nucleic acids provided by the invention comprise a complementary DNA (cDNA) copy of the corresponding mRNA transcribed in vivo from the catecholamine receptor genes of the invention. In one preferred embodiment, the mammalian catecholamine receptor is a human catecholamine receptor. In another preferred embodiment, the mammalian catecholamine receptor is a rat (Rattus norvegicus) catecholamine receptor. Also provided are the deduced amino acid sequence of the cognate proteins of the cDNAs provided by the invention, methods of making said cognate proteins by expressing the cDNAs in cells transformed with recombinant expression constructs comprising said cDNAs, and said recombinant expression constructs and cells transformed thereby.
This invention in a first aspect provides nucleic acids, nucleic acid hybridization probes, recombinant eukaryotic expression constructs capable of expressing the catecholamine receptors of the invention in cultures of transformed cells, and such cultures of transformed eukaryotic cells that synthesize the catecholamine receptors of the invention. In another aspect, the invention provides homogeneous compositions of the catecholamine receptor proteins of the invention, and membrane preparations from cells expressing the catecholamine receptor proteins of the invention, as well as antibodies against and epitopes of the catecholamine receptor proteins of the invention. The invention in another aspect provides methods for making said homogenous preparations and membrane preparations using cells transformed with the recombinant expression constructs of the invention and expressing said catecholamine receptor proteins thereby. Methods for characterizing the receptor and biochemical properties of these receptor proteins and methods for using these proteins in the development of agents having pharmacological uses related to these receptors are also provided by the invention.
In a first aspect, the invention provides a nucleic acid having a nucleotide sequence encoding a mammalian catecholamine receptor. In a first preferred embodiment, the nucleic acid encodes a human catecholamine receptor. In this embodiment of the invention, the nucleotide sequence comprises 1125 nucleotides of human catecholamine receptor cDNA comprising 1040 nucleotides of coding sequence, 20 nucleotides of 5xe2x80x2 untranslated sequence and 85 nucleotides of 3xe2x80x2 untranslated sequence. In this embodiment of the invention, the nucleotide sequence of the catecholamine receptor is the nucleotide sequence depicted in FIG. 1 (SEQ ID No:1). The sequence shown in FIG. 1 will be understood to represent one specific embodiment of a multiplicity of nucleotide sequences that encode the human catecholamine receptor amino acid sequence (SEQ ID No.: 2) of the invention and that these different nucleotide sequences are functionally equivalent and are intended to be encompassed by the claimed invention. In addition, it will be understood that different organisms and cells derived therefrom express preferentially certain tRNAs corresponding to subsets of the degenerate collection of tRNAs capable of encoding certain of the naturally-occurring amino acids, and that embodiments of the multiplicity of nucleotide sequences encoding the amino acid sequence of the human catecholamine receptor protein of the invention that are optimized for expression in specific prokaryotic and eukaryotic cells are also encompassed by the claimed invention. Isolated nucleic acid derived from human genomic DNA and isolated by conventional methods using the human cDNA provided by the invention is also within the scope of the claimed invention. Finally, it will be understood that allelic variations of the human catecholamine receptor, including naturally occurring and in vitro modifications thereof are within the scope of this invention. Each such variant will be understood to have essentially the same amino acid sequence as the sequence of the human catecholamine receptor disclosed herein.
In a second preferred embodiment of this aspect of the invention, the nucleic acid encodes the rat catecholamine receptor. In this embodiment of the invention, the nucleotide sequence includes 999 nucleotides of the rat catecholamine receptor cDNA comprising the coding sequence. In this embodiment of the invention, the nucleotide sequence of the catecholamine receptor is the nucleotide sequence depicted in FIG. 2 (SEQ ID No:3). The sequence shown in FIG. 2 will be understood to represent one specific embodiment of a multiplicity of nucleotide sequences that encode the rat catecholamine receptor amino acid sequence (SEQ ID No.: 4) of the invention and that these different nucleotide sequences are functionally equivalent and are intended to be encompassed by the claimed invention. In addition, it will be understood that different organisms and cells derived therefrom express preferentially certain tRNAs corresponding to subsets of the degenerate collection of tRNAs capable of encoding certain of the naturally-occurring amino acids, and that embodiments of the multiplicity of nucleotide sequences encoding the amino acid sequence of the rat catecholamine receptor protein of the invention that are optimized for expression in specific prokaryotic and eukaryotic cells are also encompassed by the claimed invention. Isolated nucleic acid derived from rat genomic DNA and isolated by conventional methods using the rat cDNA provided by the invention is also within the scope of the claimed invention. Finally, it will be understood that allelic variations of the rat catecholamine receptor, including naturally occurring and in vitro modifications thereof are within the scope of this invention. Each such variant will be understood to have essentially the same amino acid sequence as the sequence of the human catecholamine receptor disclosed herein.
Mammalian catecholamine receptor proteins corresponding to the human and rat cDNAs of the invention are a second aspect of the claimed invention. In a first embodiment, the mammalian catecholamine receptor protein is a human catecholamine receptor having a deduced amino acid sequence shown in FIG. 1 (SEQ ID No.:2). In a second embodiment is provided said human catecholamine receptor protein comprising a membrane preparation from a cell, most preferably a recombinant cell, expressing a nucleic acid encoding a human catecholamine of the invention. In a third embodiment, the mammalian catecholamine receptor protein is a rat catecholamine receptor having a deduced amino acid sequence shown in FIG. 2 (SEQ ID No.:4). In a fourth embodiment is provided said rat catecholamine receptor protein comprising a membrane preparation from a cell, most preferably a recombinant cell, expressing a nucleic acid encoding a rat catecholamine of the invention.
As provided in this aspect of the invention is a homogeneous composition of a mammalian catecholamine receptor having a molecular weight of about 39 kD or derivative thereof that is a human catecholamine receptor having an amino acid sequence shown in FIG. 1 and identified by SEQ ID No.:2, said size being understood to be the predicted size of the protein before any post-translational modifications thereof. Also provided is a homogeneous composition of a mammalian catecholamine receptor having a molecular weight of about 38 kD or derivative thereof that is a rat catecholamine receptor having an amino acid sequence shown in FIG. 2 and identified by SEQ ID No.:4, said size being understood to be the predicted size of the protein before any post-translational modifications thereof.
This invention provides both nucleotide and amino acid probes derived from the sequences herein provided. The invention includes probes isolated from either cDNA or genomic DNA, as well as probes made synthetically with the sequence information derived therefrom. The invention specifically includes but is not limited to oligonucleotide, nick-translated, random primed, or in vitro amplified probes made using cDNA or genomic clone of the invention encoding a mammalian catecholamine receptor or fragment thereof, and oligonucleotide and other synthetic probes synthesized chemically using the nucleotide sequence information of cDNA or genomic clone embodiments of the invention.
It is a further object of this invention to provide such nucleic acid hybridization probes to determine the pattern, amount and extent of expression of the catecholamine receptor gene in various tissues of mammals, including humans. It is also an object of the present invention to provide nucleic acid hybridization probes derived from the sequences of mammalian catecholamine receptor genes of the invention to be used for the detection and diagnosis of genetic diseases. It is an object of this invention to provide nucleic acid hybridization probes derived from the nucleic acid sequences of the mammalian catecholamine receptor genes herein disclosed to be used for the detection of novel related receptor genes.
The present invention also includes synthetic peptides made using the nucleotide sequence information comprising the cDNA embodiments of the invention. The invention includes either naturally occurring or synthetic peptides which may be used as antigens for the production of catecholamine receptor-specific antibodies, or useful as competitors of catecholamine receptor molecules for agonist, antagonist or drug binding, or to be used for the production of inhibitors of the binding of agonists or antagonists or analogues thereof to such catecholamine receptor molecules.
The present invention also provides antibodies against and epitopes of the mammalian catecholamine receptormolecules of the invention. It is an object of thepresent invention to provide antibodies that are immunologically reactive to the catecholamine receptors of the invention. It is a particular object to provide monoclonal antibodies against these catecholamine receptors. Hybridoma cell lines producing such antibodies are also objects of the invention. It is envisioned at such hybridoma cell lines may be produced as the result of fusion between a non-immunoglobulin producing mouse myeloma cell line and spleen cells derived from a mouse immunized with a cell line which expresses antigens or epitopes of a mammalian catecholamine receptor of the invention. The present invention also provides hybridoma cell lines that produce such antibodies, and can be injected into a living mouse to provide an ascites fluid from the mouse that is comprised of such antibodies. It is a further object of the invention to provide immunologically-active epitopes of the mammalian catecholamine receptorproteins of the invention. Chimeric antibodies immunologically reactive against the catecholamine receptor proteins of the invention are also within the scope of this invention.
The present invention provides recombinant expression constructs comprising a nucleic acid encoding a mammalian catecholamine receptor of the invention wherein the construct is capable of expressing the encoded catecholamine receptor in cultures of cells transformed with the construct. A preferred embodiment of such constructs comprises a human catecholamine receptor cDNA depicted in FIG. 1 (SEQ ID No.:1), such constructs being capable of expressing the human catecholamine receptor encoded therein in cells transformed with the construct. Another preferred embodiment of such constructs comprises a rat catecholamine receptor cDNA depicted in FIG. 2 (SEQ ID No.:3), such constructs being capable of expressing the human catecholamine receptor encoded therein in cells transformed with the construct.
The invention also provides prokaryotic and more preferably eukaryotic cells transformed with the recombinant expression constructs of the invention, each such cells being capable of and indeed expressing the mammalian catecholamine receptor encoded in the transforming construct, as well as methods for preparing mammalian catecholamine receptor proteins using said transformed cells.
The present invention also includes within its scope protein preparations of prokaryotic and eukaryotic cell membranes containing the catecholamine receptor protein of the invention, derived from cultures of prokaryotic or eukaryotic cells, respectively, transformed with the recombinant expression constructs of the invention.
The invention also provides methods for screening compounds for their ability to inhibit, facilitate or modulate the biochemical activity of the mammalian catecholamine receptor molecules of the invention, for use in the in vitro screening of novel agonist and antagonist compounds. In preferred embodiments, cells transformed with a recombinant expression construct of the invention are contacted with such a compound, and the binding capacity of the compounds, as well as the effect of the compound on binding of other, known catecholamine receptor agonists and antagonists, is assayed. Additional preferred embodiments comprise quantitative analyses of such effects.
The present invention is also useful for the detection of analogues, agonists or antagonists, known or unknown, of the mammalian catecholamine receptors of the invention, either naturally occurring or embodied as a drug. In preferred embodiments, such analogues, agonists or antagonists may be detected in blood, saliva, semen, cerebrospinal fluid, plasma, lymph, or any other bodily fluid.
Specific preferred embodiments of the present invention will become evident from the following more detailed description of certain preferred embodiments and the claims.